This invention relates to the field of reagentless spectrophotometric measurements of analytes in biological and non-biological samples. More specifically, the invention relates to the calibration and monitoring of calibration algorithm(s) of spectrophotometric apparatus used for analyte measurements.
Clinical laboratory tests are routinely performed on the serum or plasma of whole blood. In a routine assay, red blood cells (RBC) are separated from plasma by centrifugation, or RBC""s and various plasma proteins are separated from serum by clotting prior to centrifugation. Hemoglobin (Hb), light-scattering substances like lipid particles, and bile pigments bilirubin (BR) and biliverdin (BV) are typical blood components which will interfere with and affect spectrophotometric and other blood analytical measurements of blood analytes. Such components are referred to as interferents, and they can be measured by spectrophotometric methods. The presence of such interferents affects the ability to perform tests on the serum or plasma and as such can be said to compromise sample integrity.
Spectrophotometric measurements of blood analytes require proper calibration which can be monitored using quality control materials (QCM). QCM for blood analysis have been described in the prior art. For example U.S. Pat. No. 4,116,336 discloses the use of Amaranth or Ponceau 4 R as calibrators that mimic Hb, but these liquids must be enclosed in a flexible gas-tight container, at sub-atmospheric pressure. There is no teaching that this method is effective under atmospheric conditions. Furthermore, Amaranth and Ponceau 4 R were used separately to mimic Hb.
European Patent No. 0132399 suggests the use of one or more dyes (Acid Rhodamine B, Levafix Brilliant Yellow E-3G and Phloxine B; Phloxine Rhodamine and Atanyl Yellow 4NGL), which mimic the spectral response of whole blood and various levels of Hb in whole blood. There is no teaching of any substance or substances used to mimic an indicator of hemolysis or any analyte in serum or plasma.
WO 87/06343 discloses the use of a combination of Acid Red Dye #27 (CI 16185) and Acid Blue Dye #9 (CI 42090) and also a combination of Ponceau 3R Red Dye (CI 16155) and Acid Blue Dye #9 that simulate samples of whole blood having various levels of the fractions of Hb and of total Hb. There is no teaching of any substance or substances used to simulate an indicator of hemolysis or any analyte in serum or plasma.
Despite the fact that a number of QCM have been identified for various blood components, QCM for reagentless methods for measuring other components such as BR, BV, IL etc. are still needed. Furthermore no reliable method for selecting QCM has been described.
Warren (2001, Clinical Chemistry, Vol 47, No. 6, Supplement 2001) discloses the use of a serum pool for estimating the precision of several calibration algorithms. The use of the serum pool in monitoring the calibration algorithms is not taught.
WO-98/39634 and U.S. Pat. No. 6,268,910 B1 and U.S. Pat. No. 5,846,492 disclose methods for measuring Hb, IL, BR and BV in the presence of Hb, Hb-based blood substitute, IL, BR and BV, methods for measuring Hb-based blood substitute in the presence of Hb, IL, BR and BV, and methods for measuring Hb, IL, BR, BV and MB in the presence of Hb, IL, BR, BV and MB. However, they do not discuss QCM for monitoring calibration for any of the analytes.
It is an object of the present invention to overcome disadvantages of the prior art. This object is met by a combination of the features of the main claims. The sub-claims disclose further advantageous embodiments of the invention.
This invention relates to the field of reagentless spectrophotometric measurements of analytes. More specifically, the invention relates to the calibration and monitoring of calibration algorithm(s) of spectrophotometric apparatus used for analyte measurements.
The present invention relates to a method (method A) of monitoring calibration of a spectrophotometirc apparatus comprising one or more calibration algorithms for one or more analytes comprising:
i) measuring absorbance of a quality control material with the apparatus to obtain a measurement, the quality control material exhibiting an absorbance spectra characterized as having a negative slope for a continuous spectral segment from about 5 nm to about 200 nm in length, the spectral segment including a principal calibration wavelength for the one or more analytes;
ii) calculating one or more concentration values from the measurement using the one or more calibration algorithms;
iii) comparing the one or more concentration values with an assigned value given to the quality control material for each of the one or more analytes; and
iv) determining if there is a violation of a pre-established quality control rule, thereby monitoring the one or more calibration algorithms of the apparatus
The present invention also provides the method as just defined (method A), wherein the one or more analytes is one or more analytes in a biological fluid selected from the group consisting of serum, plasma, urine, synovial fluid and cerebrospinal fluid. If the one or more analytes is:
bilirubin, then in the step of measuring (step i)) the spectral segment is selected from wavelengths of the absorbance spectra of from about 450 nm to about 600 nm;
an indicator of hemolysis, then in the step of measuring (step i)) the spectral segment is selected from wavelengths of the absorbance spectra of from about 550 nm to about 650 nm, and the indicator of hemolysis selected from the group consisting of total Hb, Oxy-Hb, and xe2x80x9ctotal Hb minus met-Hbxe2x80x9d;
a hemoglobin-based blood substitute, then in the step of measuring (step i)) the spectral segment is selected from wavelengths of the absorbance spectra of from about 550 nm to about 700 nm;
met-hemoglobin, then in the step of measuring (step i)) the spectral segment is selected from wavelengths of the absorbance spectra of from about 610 nm to about 690 nm;
methylene blue, the in the step of measuring (step i)) the spectral segment is selected from wavelengths of the absorbance spectra of from about 650 nm to about 750 nm;
biliverdin, the in the step of measuring (step i)) the spectral segment is selected from wavelengths of the absorbance spectra of from about 650 run to about 800 nm.
The present invention also pertains to the method as just described (method A) wherein the quality control material comprises one or more substances selected from the group consisting of a dye, copper sulfate, total Hb, Oxy-Hb, carboxy-Hb, xe2x80x9ctotal Hb minus met-Hbxe2x80x9d, cyanmet-Hb, a Hb-based blood substitute, Intralipid and a perflurocarbon-like blood substitute. Furthermore, the absorbance spectra of the one or more substances may be altered by adding a modifier. Preferably the modifier causes a spectral shift in the absorbance spectra. The modifer may be selected from the group consisting of a polymer, a protein, amaranth, and a combination thereof.
The present invention also relates to a method of monitoring calibration of a spectrophotometric apparatus comprising one or more calibration algorithms for a perflurocarbon-like blood substitute, turbidity, or a combination thereof, comprising:
i) measuring absorbance of a quality control material with the apparatus to obtain a measurement, the quality control material exhibiting an absorbance spectra from about 700 nm to about 1100 nm;
ii) calculating one or more concentration values from the measurement using the one or more calibration algorithms;
iii) comparing the one or more concentration values with an assigned value given to the quality control material for each of the perflurocarbon-like blood substitute, turbidity, or a combination thereof; and
iv) determining if there is a violation of a pre-established quality control rule, thereby monitoring the one or more calibration algorithms of the apparatus
The present invention also provides a method (method B) for determining the concentration of one or more analytes in a sample in a spectrophotometirc apparatus comprising at least one primary calibration algorithm comprising:
i) monitoring calibration of the apparatus as defined above (method A);
ii) establishing that there is no violation of a pre-established quality control rule:
iii) measuring absorbance values of the sample;
iv) calculating an order derivative of absorbance of the sample; and
v) determining a concentration of the one or more analytes in the sample, by applying the primary calibration algorithm to the order derivative of absorbance value.
The present invention relates to the method as just defined (method B), wherein the one or more analytes is one or more blood analytes. If the one or more blood analytes is:
bilirubin, then in the step of measuring (step I), Method A) the spectral segment is selected from wavelengths of the absorbance spectra of from about 450 nm to about 600 nm;
an indicator of hemolysis, then in the step of measuring (step i), Method A) the spectral segment is selected from wavelengths of the absorbance spectra of from about 550 nm to about 650 nm, and the indicator of hemolysis selected from the group consisting of total Hb, Oxy-Hb, and xe2x80x9ctotal Hb minus met-Hbxe2x80x9d;
a hemoglobin-based blood substitute, then in the step of measuring (step i), Method A) the spectral segment is selected from wavelengths of the absorbance spectra of from about 550 nm to about 700 nm;
met-hemoglobin, then in the step of measuring (step i), Method A) the spectral segment is selected from wavelengths of the absorbance spectra of from about 610 nm to about 690 nm;
methylene blue, the in the step of measuring (step i), Method A) the spectral segment is selected from wavelengths of the absorbance spectra of from about 650 nm to about 750 nm;
biliverdin, the in the step of measuring (step i), Method A) the spectral segment is selected from wavelengths of the absorbance spectra of from about 650 nm to about 800 nm.
The present invention also pertains to the method as just described (method B) wherein the quality control material comprises one or more substances selected from the group consisting of a dye, copper sulfate, total Hb, Oxy-Hb, carboxy-Hb, xe2x80x9ctotal Hb minus met-Hbxe2x80x9d, cyanmet-Hb, a Hb-based blood substitute, Intralipid and a perflurocarbon-like blood substitute. Furthermore, the absorbance spectra of the one or more substances may be altered by adding a modifier. Preferably the modifier causes a spectral shift in the absorbance spectra. The modifer may be selected from the group consisting of a polymer, a protein, amaranth, and a combination thereof.
Furthermore, the present invention relates to method for determining the concentration of one or more analytes in a sample in a spectrophotometric apparatus comprising at least one primary calibration algorithm comprising:
i) monitoring calibration of the apparatus as defined in Method A;
ii) establishing that there is no violation of a pre-established quality control rule:
iii) measuring absorbance values of the sample;
iv) calculating an order derivative of absorbance of the sample; and
v) calculating a concentration of a perflurocarbon-like blood substitute, turbidity, or a combination thereof in terms of a lipid emulsion in the sample, by applying said primary calibration algorithm to said order derivative of absorbance value.
In one aspect of the invention there is provided method (Method C) for selecting one or more substances as a quality control material for monitoring at least one primary calibration algorithm on an apparatus comprising:
i) identifying a principal calibration wavelength for each of one or more of an analyte;
ii) screening absorption spectra of the one or more substances; and
iii) selecting one or more of the substances exhibiting a negative slope of the absorbance spectra, for a continuous spectral segment from about 5 nm to about 200 nm, the spectral segment including the principal calibration wavelength.
The present invention relates to the method as just defined (method C), wherein the one or more of an analyte is one or more analyte in a biological fluid selected from the group consisting of serum, plasma, urine, synovial fluid and cerebrospinal fluidis. If the one or more analyte is:
bilirubin, then in the step of (selecting, step iii)) the spectral segment is selected from wavelengths of the absorbance spectra of from about 450 nm to about 600 nm;
an indicator of hemolysis, then in the step of (selecting, step iii)), the spectral segment is selected from wavelengths of the absorbance spectra of from about 550 nm to about 650 nm, and the indicator of hemolysis selected from the group consisting of total Hb, Oxy-Hb, and xe2x80x9ctotal Hb minus met-Hbxe2x80x9d;
a hemoglobin-based blood substitute, then in the step of (selecting, step iii)), the spectral segment is selected from wavelengths of the absorbance spectra of from about 550 nm to about 700 nm;
met-hemoglobin, then in the step of (selecting, step iii)), the spectral segment is selected from wavelengths of the absorbance spectra of from about 610 nm to about 690 nm;
methylene blue, the in the step of (selecting, step iii)), the spectral segment is selected from wavelengths of the absorbance spectra of from about 650 nm to about 750 nm;
biliverdin, the in the step of (selecting, step iii)), the spectral segment is selected from wavelengths of the absorbance spectra of from about 650 nm to about 800 nm.
The present invention also pertains to the method as just described (method C) wherein the one or more substances is selected from the group consisting of a dye, copper sulfate, total Hb, Oxy-Hb, carboxy-Hb, xe2x80x9ctotal Hb minus met-Hbxe2x80x9d, cyanmet-Hb, a Hb-based blood substitute, Intralipid and a perflurocarbon-like blood substitute. Furthermore, the absorbance spectra of the one or more substances may be altered by adding a modifier. Preferably the modifier causes a spectral shift in the absorbance spectra. The modifer may be selected from the group consisting of a polymer, a protein, amaranth, and a combination thereof.
Also provided in the present invention is a method for selecting one or more substances as a quality control material for monitoring at least one primary calibration algorithm on a spectrophotometric apparatus for one or more of a perflurocarbon-like blood substitute and turbidity, wherein turbidity is measured in terms of concentration units of a lipid emulsion, comprising:
i) identifying a principal calibration wavelength for each of one or more of of the perflurocarbon-like blood substitute and the turbidity;
ii) screening absorption spectra of the one or more substances; and
iii) selecting one or more of the substances exhibiting absorbance within the range from about 700 nm to about 1100 nm.
Furthermore the present invention pertains to the method as just defioned wherein the step of selecting (step iii)) is replaced within the following step of selecting:
iii) selecting one or more of the substances exhibiting absorbance spectra as having a negative slope for a continuous spectral segment from about 5 nm to about 400 nm within the range of wavelengths from about 700 nm to about 1100 nm.
The present invention provides a method (D) of monitoring the calibration of a reagentless spectrophotometric apparatus comprising one or more calibration algorithms for one or more analytes in a sample, comprising:
i) measuring absorbance of a quality control material with the spectrophotometric apparatus to obtain one or more measurements, the quality control material comprising one or more substances that absorb electromagnetic radiation, whereby predicted values for the one or more analytes can be obtained;
ii) calculating one or more of the predicted values from the one or more measurements;
iii) comparing the one or more of the predicted values with one or more assigned values given to the quality control material for the one or more analytes; and
iv) determining if there is a violation of a pre-established quality control rule, thereby monitoring the calibration algorithms of the spectrophotometric apparatus.
Furthermore, the step of measuring (step i)) can be performed in any transparent or translucent vessel, and wherein the one or more wavelengths used in the one or more calibration algorithms is selected from the range from about 450 nm to 3000 nm.
The present invention also pertains to method (D) wherein the sample is a biological fluid, non-biological fluid, semi-solid, or a soft solid, and wherein the one or more calibration algorithms are developed using a statistical technique selected from the group consisting of simple linear regression, multiple linear regression, partial least squares and principal component analysis. Also included in the present invention, is the method (D), wherein the one or more analytes are selected from the group consisting of a simulator of turbidity, a perfluorocarbon-like blood substitute, bilirubin, an indicator of hemolysis, a Hb-based blood substitute, methylene blue, met-Hb and biliverdin, and the sample is selected from the group consisting of whole blood, serum, plasma, urine, synovial fluid and cerebrospinal fluid.
In yet another aspect of the invention there is provided a quality control material for mimicking two or more analytes comprising, one or more substances having a combined absorption spectrum exhibiting a negative slope for a continuous spectral segment from about 5 nm to 200 nm in a portion of an absorption spectrum, including a principal calibration wavelength, for the two or more analytes. The present invention also provides a quality control material as just described, wherein the two or more analytes are selected from the group consisting of whole blood, serum, plasma, synovial fluid, cerebrospinal fluid, urine, mucus, lymphatic fluid, semen and feces, and wherein one of the two or more analytes is:
bilirubin, and the spectral segment is selected from wavelengths of absorbance spectra of from about 450 nm to about 600 nm;
an indicator of hemolysis, and the spectral segment is selected from wavelengths of absorbance spectra of from about 550 m to about 650 nm, indicator of hemolysis selected from the group consisting of total Hb, Oxy-Hb, and xe2x80x9ctotal Hb minus met-Hbxe2x80x9d;
hemoglobin-based blood substitute, and the spectral segment is selected from wavelengths of the absorbance spectra of from about 550 nm to about 700 nm;
met-hemoglobin, and the spectral segment is selected from wavelengths of the absorbance spectra of from about 610 nm to about 690 nm;
methylene blue, and the spectral segment is selected from wavelengths of the absorbance spectra of from about 650 nm to about 750 nm;
biliverdin, and the spectral segment is selected from wavelengths of the absorbance spectra of from about 650 nm to about 800 nm;
either a simulator of turbidity, or a perflurocarbon-like blood substitute characterized as having an absorbance spectra of from about 700 nm to about 1100 nm;
either a simulator of turbidity or a perflurocarbon-like blood substitute, characterized as having a negative slope for a continuous spectral segment from about 5 nm to about 400 nm within the range of from about 700 nm to about 1100 nm.
The quality control material as just described may also comprise a dye, copper sulfate, total Hb, Oxy-Hb, carboxy-Hb, xe2x80x9ctotal Hb minus met-Hbxe2x80x9d, cyanmet-Hb, a Hb-based blood substitute, a lipid emulsion and a perflurocarbon-like blood substitute, or a combination thereof. Furthermore, the absorbance spectra of the quality control material may be altered by adding a modifier. Preferably the modifier causes a spectral shift in the absorbance spectra. The modifer may be selected from the group consisting of a polymer, a protein, amaranth, and a combination thereof.
The present invention embraces a quality control material for use in a reagentless spectrophotometric apparatus, comprising, one or more substances that mimic one or more analytes in serum, plasma, urine, synovial fluid or cerebrospinal fluid. The one or more analytes of the quality control material may be selected from the group consisting of a simulator of turbidity, a perfluorocarbon-like blood substitute, bilirubin, an indicator of hemolysis, a Hb-based blood substitute, methylene blue, met-Hb and biliverdin. When the one or more analytes is an indicator of hemolysis in serum or plasma, the indicator of hemolysis is one of total Hb, oxy-Hb or xe2x80x9ctotal Hb minus met-Hb,xe2x80x9d and the quality control material is exposed to atmospheric conditions. The present invention pertains to the quality control material as just above, wherein the one or more substances are selected from the group consisting of amaranth, acid fuchsin, basic fuchsin, ponceau S, chromotrope 2R, phenol red, crystal ponceau, methyl orange, a Hb-based blood substitute, total Hb, oxy-Hb, carboxy-Hb, cyanmet-Hb, a polymer, and a protein. The present invention also provides a quality control material as just defined for use in a reagentless spectrophotometric apparatus, comprising, one or more substances that mimics an indicator of hemolysis, wherein the indicator of hemolysis is selected from the group consisting of oxy-Hb and xe2x80x9ctotal Hb minus met-Hb,xe2x80x9d and wherein the quality control material is exposed to atmospheric conditions. Preferably the quality control material is not supplemented with bilirubin.
The present invention pertains to a quality control material for use in a reagentless spectrophotometric apparatus, comprising, one or more substances that mimics an indicator of hemolysis, wherein the one or more substances are selected from the group consisting of total Hb, oxy-Hb, xe2x80x9ctotal Hb minus met-Hb,xe2x80x9d cyanmet-Hb, amaranth, acid fuchsin, basic fuchsin, ponceau S, chromotrope 2R, phenol red, crystal ponceau, methyl orange, a Hb-based blood substitute, oxy-Hb, carboxy-Hb, cyanmet-Hb, a polymer, and a protein., and wherein the quality control material is exposed to atmospheric conditions. Preferably the quality control material is not supplemented with bilirubin.
Also, the present invention is directed to a quality control material for use in a reagentless spectrophotometric apparatus, comprising, one or more substances that mimics an indicator of hemolysis, wherein the one or more substances are selected from the group consisting of total Hb, oxy-Hb, xe2x80x9ctotal Hb minus met-Hb,xe2x80x9d cyanmet-Hb, acid fuchsin, basic fuchsin, ponceau S, chromotrope 2R, phenol red, crystal ponceau, methyl orange, a Hb-based blood substitute, oxy-Hb, carboxy-Hb, cyanmet-Hb, a polymer, and a protein. Preferably the quality control material is not supplemented with bilirubin.
The present invention embraces a quality control material for use in a reagentless spectrophotometric apparatus, comprising, one or more substances that mimic an indicator of hemolysis, wherein the indicator of hemolysis is selected from the group consisting of oxy-Hb and xe2x80x9ctotal Hb minus met-Hbxe2x80x9d. Preferably the quality control material is not supplemented with bilirubin.
The present invention also provides a quality control material for use in a reagentless spectrophotometric apparatus, comprising, one or more substances that mimics one or more of, biliverdin, bilirubin, methelene blue, met-Hb, a simulator of turbidity, a perflurocarbon-like blood substitute, a Hb-based blood substitute.
The present invention includes a quality control material for use in a reagentless spectrophotometric apparatus, comprising, one or more substances that mimics one or more of, an indicator of hemolysis, biliverdin, bilirubin, methelene blue, met-Hb, a simulator of turbidity, a perflurocarbon-like blood substitute, a Hb-based blood substitute wherein said indicator of hemolysis is selected from the group consisting of oxy-Hb and xe2x80x9ctotal Hb minus met-Hbxe2x80x9d. Preferably the quality control material is not supplemented with bilirubin.
The present invention also pertains to a quality control material for use in a reagentless spectrophotometric apparatus, comprising, one or more substances that mimics an indicator of hemolysis, wherein the substance is selected from the group consiting of total-Hb and oxy-Hb, wherein the oxy-Hb accounts for about 95% of total Hb, or the total-Hb comprises about 95% oxy-Hb, and wherein the quality control material is exposed to atmospheric conditions. Preferably the quality control material is not supplemented with bilirubin.
The present invention also provides a method (E) for producing a corrected predicted value for an indicator of hemolysis in a sample, in the presence of met-Hb, the method comprising the steps of:
i) developing a first primary calibration algorithm for one of a total Hb, or an oxy-Hb, for predicting a first value for either the total Hb or the oxy-Hb in said sample;
ii) deriving a second primary calibration algorithm for the met-Hb, for predicting a second value for the met-Hb in the sample; and
iii) adding the predicted second value for the met-Hb to either the predicted first value for total Hb or the predicted first value for oxy-Hb, to a produce a the corrected predicted value for an indicator of hemolysis.
Furthermore, the present invention pertains to the method as just described (method (E)), wherein the step of developing (step i)) and the step of deriving (step ii)), each comprises the steps of:
a) collecting an absorbance measurement for each calibration sample in a primary calibration set, the calibration sample having known reference values for each analyte;
b) calculating an order derivative of absorbance for each of the calibration sample; and
c) creating a primary calibration algorithm for each of the indicator of hemolysis and the met-Hb using the derivative of absorbance, the known reference values, and a statistical technique.
The present invention also includes the above method (method (E)), wherein in the step of collecting (step a)), the reference values for either the total Hb or the oxy-Hb, are obtained from the measured amounts of the total Hb or the oxy-Hb, in the presence of one or more of oxy-Hb, deoxy-Hb, carboxy-Hb and met-Hb in the calibration samples. Preferably, the oxy-Hb accounts for about 95% of total Hb, or the total-Hb comprises about 95% oxy-Hb, and the sample is one of serum, plasma, urine, synovial fluid or cerebrospinal fluid.
The present invention also embraces a method (F) for flagging a predicted value for an indicator of hemolysis in a sample, in the presence of met-Hb, comprising:
i) developing a first primary calibration algorithms for one of a total Hb or an oxy-Hb, for predicting a value for either the total Hb or the oxy-Hb in said sample;
ii) deriving a second primary calibration algorithm for the met-Hb, for predicting a second value for the met-Hb in said sample;
iii) determining if the predicted met-Hb value exceeds a pre-determined value; and
iv) flagging the predicted value for the total Hb, said oxy-Hb or a combination thereof, when the predicted met-Hb value exceeds said pre-determined value.
Furthermore, the present invention provides the method (F) defined above wherein the steps of developing (step i)) and deriving (step ii)), each comprises the steps of:
a) collecting an absorbance measurement for each calibration sample in a primary calibration set, the calibration sample having known reference values for each analyte;
b) calculating an order derivative of absorbance for each of the calibration sample; and
c) creating a primary calibration algorithm for each of the indicator of hemolysis and the met-Hb using the derivative of absorbance, the known reference values, and a statistical technique.
Preferably, in the step of collecting (step a)), the reference values for each of the total Hb or the oxy-Hb are obtained from the measured amounts of the total Hb or the oxy-Hb, in the presence of one or more of oxy-Hb, deoxy-Hb, carboxy-Hb and met-Hb in the calibration samples, and wherein the oxy-Hb accounts for about 95% of total Hb, or wherein the total-Hb comprises about 95% oxy-Hb. Furthermore, the invention includes the above method wherein sample is one of serum, plasma, urine, synovial fluid or cerebrospinal fluid.
This summary does not necessarily describe all necessary features of the invention but that the invention may also reside in a sub-combination of the described features.